7. Project Summary/Abstract This F31 application is to investigate the cellular and molecular mechanisms underlying alcohol inhibition of bone fracture healing. Alcohol consumption is associated with increased risk-taking and injuries leading to a bone fracture. In addition, patients using alcohol have an increased risk of fracture nonunion, which is associated with increased healthcare costs and substantial patient morbidity. Bone fracture repair is a complex process involving the differentiation of local periosteum mesenchymal stem cells (MSC) near the injury site into chondrocytes and osteoblasts to form a fracture callus. Our laboratory has previously reported that rodents exposed to episodic alcohol preceding a midline tibia fracture develop a deficient fracture callus as characterized by a reduced cartilaginous callus volume, diameter and histological evidence of inhibited cartilage maturation. These data suggest that alcohol may be inhibiting fracture callus formation by inhibiting the differentiation of MSC to chondro- osteo lineages. Alcohol exposure is known to produce intracellular reactive oxygen species (ROS), and ROS has been shown to play an important role in cellular signaling regulating MSC self-renewal and differentiation. However, the molecular mechanism connecting ROS to chondro-osteo differentiation inhibition has yet to be described. Activation of molecular oxidative stress responder family of forkhead box O (FoxO) transcription factors have been shown to be detrimental to overall bone health, and it has been previously reported that FoxO- specific signaling has been shown to antagonize Canonical Wnt signaling activity critical for MSC chondro-osteo lineage commitment. Recently, we have reported that FoxO1/3 activation is associated with decreased callus area following fracture injury in alcohol-exposed rodents. Based on these preliminary findings, we hypothesize that alcohol-exposure inhibits MSC to chondro-osteo differentiation within the fracture callus through ROS-mediated enhanced FoxO1/3 signaling. To test this hypothesis, we propose the following aims. Aim 1 will characterize alterations in MSC chondrogenic lineage differentiation within the fracture callus of alcohol- exposed rodents. Aim 2 will determine the effect of alcohol on primary MSC differentiation and whether attenuation of MSC FoxO1/3 signaling restores MSC differentiation. Overall, we expect this study will open new avenues for therapeutic intervention to attenuate the risks associated with alcohol consumption and bone fracture repair.